Platelets of pseudohypoparathyroid patients: Evidence that distinct receptor-cyclase coupling proteins mediate stimulation and inhibition of adenylate cyclase

Abstract

We studied platelets of patients with the genetic disorder pseudohypoparathyroidism (PHP) to test whether the nucleotide-binding proteins mediating stimulation of adenylate cyclase (termed NS) are identical to those mediating inhibition of cyclase (termed Ni). Functional responses to hormones that work through stimulation of adenylate cyclase are blunted in PHP patients. The erythrocytes of many of these patients (PHP-Ia) have previously been shown to have decreased Ns activity whereas those of other PHP patients (PHP-Ib) have normal Ns activity. We find that this decreased Ns activity (measured by the ability to restore adenylate cyclase activity to membranes prepared from S49 cyccells) also occurs in the platelets of PHP-Ia but not of PHP-Ib patients. Platelets from both groups of patients accumulate less cAMP in response to prostacyclin than do platelets from control subjects. In contrast to the decreased Ns function in patients with PHP-Ia, we find that Ni function in platelets is similar in these patients and control subjects in several types of experiments: (i) epinephrine-mediated inhibition ofprostacyclin-stimulated cAMP production in intact platelets; (ii) the affinity of platelet a%-adrenergic receptors for epinephrine, as determined by competition for [3H]yohimbine binding; (iii) the decrease in receptor affinity for epinephrine produced by Na+ and GTP; and (iv) METHODSPatients. Seven patients with PHP were studied; these were the same patients reported previously (7). Previous assays demonstrated decreased activity of erythrocyte N, in three of the patients (PHP-Ia) but not in four others (PHP-Ib) (7). Control platelets were obtained from healthy men (ages 22-45) who had taken no medication for at least 1 wk.Platelets. Blood was drawn into 60-ml plastic syringes containing 6 ml of 3.8% sodium citrate and centrifuged at 300 X g for 20 min, and the platelet-rich plasma was removed. The packed erythrocytes were mixed with an equal volume of 100 mM NaCl/5 mM EDTA/50 mM Tris-HCl, pH 7.5, and were centrifuged twice. The platelet-rich plasma and the two extracts were combined and centrifuged at 5,000 X g for 10 min and the pellet was washed twice by the same procedure. Determination of cAMP accumulation and some of the radioligand binding assays were carried out with intact platelets suspended in 50 mM Tris-HCl/100 mM NaCV5 mM EDTA, pH 7.5. In other raAbbreviations: PHP, pseudohypoparathyroidism; Ni, nucleotide binding unit coupled to inhibition ofadenylate cyclase; N,, nucleotide binding unit coupled to stimulation ofadenylate cyclase; PGI2, prostaglandin I2 or prostacyclin. 4193The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Deficient guanine nucleotide regulatory unit activity in cultured fibroblast membranes from patients with pseudohypoparathyroidism type I. A cause of impaired synthesis of 3',5'-cyclic AMP by intact and broken cells

“…This component is the G unit, and it is apparent from our current results that a deficiency of G units can impair the ability of an intact cell to accumulate cAMP in response to hormone stimulation and can impair adenylate cyclase activation of membranes by agents acting via the G unit. A similar conclusion can be drawn from data recently reported by Motulsky et al (32). Although Bourne et al (23) were unable to see a significant difference in cAMP response to PGE1 in intact fibroblasts derived from control subjects vs. patients with PHP, the same investigators (32) found deficient G-unit activity in platelet membranes as well as reduced cAMP response to prostacyclin in intact platelets from patients with PHP.…”

“…A similar conclusion can be drawn from data recently reported by Motulsky et al (32). Although Bourne et al (23) were unable to see a significant difference in cAMP response to PGE1 in intact fibroblasts derived from control subjects vs. patients with PHP, the same investigators (32) found deficient G-unit activity in platelet membranes as well as reduced cAMP response to prostacyclin in intact platelets from patients with PHP.…”

Defective Stimulation of Adipocyte Adenylate Cyclase, Blunted Lipolysis, and Obesity in Pseudohypoparathyroidism 1a

“…Other reasons for their different conclusion are that their samples were stored for long periods and thcir basal FFA levels were well above the upper limit of normal values. G,-mediated events in platelets have been reported to be normal in PHPT (21). The inhibition of adipocyte adenylate cyclase via the adenosine A l receptor was normal in our patients, which is in contrast with what is usually found in obesity (4.…”

Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors.

“…Activation and inhibition of adenylate cyclase are differentially sensitive to Mn2+ (10), GTP (10)(11)(12), and chemical modification by N-ethylmaleimide (6,13). Platelets from pseudohypoparathyroid patients are functionally deficient in Ns but express both epinephrine-mediated inhibition of adenylate cyclase and GTP-sensitive high-affinity binding of agonists to a2-adrenergic receptors, suggesting that N1 is fully functional in these cells (14). Furthermore, cyc-S49 lymphoma cells lack functional Ns but express active N, (15).…”